Fluorescence microscopy is a very effective light microscopy method for locating proteins and for imaging protein distributions in tissue samples and cells (hereinafter called sample). For this purpose, the sample to be microscoped is selectively supplied with fluorescent dyes which dock to the proteins. By illumination with light (excitation light) of a specific wavelength (excitation wavelength), depending on the dye used, the latter is excited and electrons of the dye molecules (fluorophores) are raised to a higher energy level. After a short dwell time, the excited electrons return to their original level, each emitting light (emission light) of a specific wavelength (emission wavelength), which is longer than the excitation wavelength. In this case, a single electron emits a light quantum or photon. This property is used to visualize proteins or other substances.
Fluorescence microscopes, unless they serve exclusively for visual examination of the sample, are divided into classic wide-field microscopes, with a camera comprising a plurality of detectors arranged in a matrix, and into laser-scanning microscopes comprising only one single detector. Both types of microscopes are common nowadays, with the laser-scanning microscopes having many advantages over the classic wide-field fluorescence microscopes.
Laser-scanning microscopes are usually confocal microscopes, which means the focus of the excitation light, which is placed in the object plane, is imaged into a conjugated plane, i.e. the image plane, where a single detector is positioned, usually with a pinhole in front of it. In this manner, only emission light of a tiny object field portion, i.e. of a tiny pixel, is directed onto the detector. This has the decisive advantage over the classic wide-field fluorescence microscope that only light quantums of the emission light coming from the focal plane are detected and contribute to signal formation. In this manner, samples can be microscoped layer by layer, and the fluorescent images generated from the signals for each microscoped layer can be combined into a three-dimensional image.
The fluorescent images per layer are obtained by the object field being scanned by a laser scanning system, usually consisting of two galvanometric mirrors, at a scanning frequency resulting in a pixel dwell time per pixel. The emission light received by the detector during the pixel dwell time is converted by the detector into electrical analog detector signals which, in knowledge of the respective position of the laser scanning system, are each associated with a respective pixel. At first, the analog detector signals are digitized at a predetermined clock frequency. For this purpose, two types of digitization are basically known in the field of laser scanning microscopy.
A first type of digitization is the integration via the analog detector signal. It is used when the duration of the signal detection (detection time) is of the same magnitude as the pixel dwell time, i.e. the detection time is only marginally shorter than the pixel dwell time. Based on the integral value formed via the analog signal in this case and the detection time, one mean value per pixel (mean amplitude) can be derived from one single readout of the detector, representing a total amplitude associated with the pixel.
In the past, due to the low radiation intensity of the fluorescent dyes, particular use was made of this integration method, because it allows a comparatively high total amplitude to be formed.
A second type of digitization is the fast sampling of the analog detector signal. It is preferably used if the detection time is considerably shorter, in particular several orders of magnitude shorter than the pixel dwell time. Via the pixel dwell time, a respective amplitude is generated for each of a plurality of measuring times (the duration of signal detection being simply regarded as a point in time, or the measuring time being the time at which the detector is read out) and a mean value (mean amplitude) is formed from the sequence of amplitudes and assigned as the total amplitude to one respective pixel.
Today, this fast sampling is common in laser scanning microscopy because, when using modern A/D converters, the advantages of the method prevail.
Typically, the detector signal is converted at a constant sampling rate which is considerably, preferably at least one order of magnitude, in particular two orders of magnitude, higher than the shortest possible pixel dwell time. The number of measuring times per pixel is then obtained from the pixel dwell time. The latter is in turn defined by the scanning setting (frame rate, pixel number, scan size). An increase in pixel dwell time then results in an increase in the number of measuring times per pixel.
In order to improve the signal-to-noise ratio, the pixel dwell time is usually increased. This has the disadvantage of increasing the total time for obtaining a fluorescent image formed from the total amplitudes for all pixels in the form of raster graphics and of decreasing the frame rate. The signal-to-noise ratio is determined by the ratio of the amounts of the total amplitude which are caused, on the one hand, by the emission light and, on the other hand, by the noise.